Materials Transactions, Vol. 46, No. 7 (2005) pp.

1610 to 1617
Special Issue on Medical, Healthcare, Sport and Leisure Materials
#2005 The Japan Institute of Metals

Osteocompatibility of Stainless Steel, CoCrMo, Ti6Al4V

and Ti15Zr4Nb4Ta Alloy Implants in Rat Bone Tissue
Yoshimitsu Okazaki1 , Emiko Gotoh2 , Miki Nishimori3 , Shin-ichi Katsuda3 ,
Takeshi Manabe4 and Kihei Kobayashi4
1
Institute for Human Science and Biomedical Engineering, National Institute of Advanced Industrial Science and Technology,
Tsukuba 305-8564, Japan
2
National Institute of Technology and Evaluation, Tsukuba 305-0044, Japan
3
Japan Food Research Laboratory, Tokyo 206-0025, Japan
4
Nihon University of Dentistry at Matsudo, Matsudo 271-8587, Japan

To examine the formation of a new bone using various metal implants, 316L stainless steel, CoCrMo casting alloy, and Ti6Al4V and
Ti15Zr4Nb4Ta alloys were implanted into the rat femur and tibia for up to 48 weeks. Morphometrical parameters, namely, new bone
formation rate, bone contact rate, new bone thickness and osteoid formation rate were investigated. Although a thin osteoid layer in a new bonemetal interface was observed in all the alloy implants, a new bone was well formed around all the alloy implants in the bone marrow of the rat
femur and tibia up to 48 weeks. Neither the resorption of bone nor inammatory reactions such as the presence of foreign-body giant cells and
inltration of inammatory cells were also evident in the histological examination of these implants. A normal bone remodeling was observed in
the new bone-metal implant interface, and osteoblasts capable of dierentiating into a new bone tissue were lined on the implant side in the new
bone-metal implant interface. Many osteocytes were observed in the lamellar bone tissue. The new bone formed around all the alloy implants
developed into a calcied bone consisting of lamellar structure with increasing implantation period. A capsulated brous connective tissue was
observed in the 316L stainless steel and CoCrMo alloy implants at 48 weeks after long-term implantation. Many osteoclasts were observed at
the interface between the brous connective tissue and lamellar bone tissue. The bone formation rates around all the alloy implants were
markedly high, approximately more than 90% at 4 weeks after implantation, and thereafter, no marked change was observed. The bone contact
rate of CoCrMo alloy implant was slightly higher than that of 316L stainless steel implant. In the early stage of implantation (412 weeks), the
bone contact rates of Ti alloy implants were signicantly higher than that of CoCrMo alloy implant. In the late stage of implantation (2448
weeks), the osteoid formation rates of CoCrMo and Ti6Al4V alloy implants tended to increase, but not signicantly. Signicant dierences
in the bone morphometrical parameters, suggesting osteocompatibility, were detected, although histological ndings were not evident.
Histological examinations of undecalcied sections were essential for conrming the interface between a newly formed bone and a metal
implant, and morphometrical parameters, suggested to be good markers of osteocompatibility for the investigation of various metal implants.
(Received January 20, 2005; Accepted March 11, 2005; Published July 15, 2005)
Keywords: In vivo test, rat implantation, stainless steel, cobalt alloy, titanium alloy, osteocompatibility, morphometrical parameters, bone
formation, bone contact rate, new bone thickness, osteoid formation rate

1.

Introduction

Stainless steel, CoCrMo alloy and Ti alloy have been

widely used in medical implants. Osteocompatibility, osteoconduction and osteoinduction are essential properties of
metal implants used as articial joints and articial tooth
roots. Over the last few decades, the osteocompatibility of the
metal implants has been the subject of controversy.15)
However, there is still room for further investigation
involving histological analysis and histomorphometrical
parameters for osteocompatibility.15) The purpose of this
study is to histologically examine the formation of a new
bone and morphometrical parameters of osteocompatibility
for various metal implants. 316L stainless steel, CoCrMo
casting alloy, and Ti6Al4V and Ti15Zr4Nb4Ta alloys
were implanted into the rat femur and tibia for up to 48
weeks. The new bone formation rate, bone contact rate, new
bone thickness and osteiod formation rate were measured
using undecalcied bone sections. In addition to the
histological examination, quantitative analyses were performed to compare the properties of various alloy implants.

Standard (JIS) G 4303 was prepared by vacuum-induction

melting. After soaking at 1200
C for 3 h, the ingot was
forged. The billet soaked at 1200
C for 1 h was hot-worked.
After maintaining at 1050
C for 30 min, the plate was
quenched in water. Finally, the stainless steel plate was
solution-treated at 1050
C for 2 min, and then quenched in
water. The CoCrMo alloy specied in ISO 5832-4 was
subjected to vacuum-induction melting, and then vacuumcast by pouring at 1420
C into a mold manufactured using a
lost wax process. The ingot was homogenized at 1220
C for
4 h. The CoCrMo alloy was used as a cast in an in vivo test.
The Ti6Al4V extra low interstitial (ELI) alloy (ISO 58323) and the Ti15Zr4Nb4Ta alloy specied for surgical
implants in JIS T 7401-4 were subjected to vacuum-arc
melting. After
(after soaking: 1150
C3 h for Ti6Al4V
and 1050
C4 h for Ti15Zr4Nb4Ta) and 
forging
(starting temperatures: 930
C for Ti6Al4V and 750
C for
Ti15Zr4Nb4Ta), the Ti alloys were annealed for 2 h at
700
C. The chemical compositions of stainless steel and Co
CrMo and Ti alloys are shown in Table 1.
2.2

2.

Experimental Methods

2.1 Alloy specimens

316L stainless steel, which is used for the manufacture of
surgical implants in Japan, specied in Japanese Industrial

Rat implantation and preparation of undecalcied

or decalcied section
Implant specimens, 1.2 mm in diameter and 2 mm in
length, were cut from the alloy specimens. The edges of the
column were then chamfered (C 0.1). The means and
standard deviations of surface roughness (Ra) for 316L

Osteocompatibility of Stainless Steel, CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta Alloy Implants in Rat Bone Tissue
Table 1

Chemical composition (mass %) of materials used.

Cr

Ni

Mo

Mn

316L stainless steel

17.00

12.15

2.04

1.40

CoCrMo alloy

28.36

0.3

6.1

0.42

Titanium alloys
Ti15Zr4Nb4Ta
Ti6Al4V ELI

1611

Si

Fe

Co

0.024

0.82

0.034

0.008

Bal.

0.27

0.63

0.46

Bal.

Zr

Nb

Ta

Pd

Fe

Al

Ti

15.24

3.90

3.92

0.22

0.022
0.179

0.162
0.174

0.048
0.0032

0.011

0.002
0.007

6.24

4.19

Bal.
Bal.

stainless steel, CoCrMo, Ti6Al4V and Ti15Zr4Nb

4Ta alloy implant specimens were 0:11  0:03, 1:32  0:42,
1:36  0:18, and 0:77  0:07, respectively. The surface
roughness was measured at two sites of the three specimens
for each alloy implant.
The implantation test was also performed in reference to
ISO 10993-6 (Biological evaluation of medical devices-part
6: Test for local eects after implantation). In this study,
implantation tests were performed at two laboratories,
namely, the Nihon University School of Dentistry at Matsudo
and the Japan Food Research Laboratory.
The experimental protocol was approved by the Laboratory Animal Ethics Committee of the Nihon University
School of Dentistry at Matsudo. Six-week-old male Wistar
rats (purchased from Sankyo Labo Service) weighing
184  30 g after more than 1 week of feeding were selected
because it is at this growth period where bone formation is
promoted around an implant. After pentobarbital sodium
solution was intra-abdominally injected at a dose of 25 mg/
kg, two sites 20 mm below the knee joints on the right and left
sides were shaved, sterilized and incised using a scalpel to
expose the surface of tibia and exfoliate the periosteum. For
the implant cavity, two holes 1.2 mm in diameter were
formed at 8 and 15 mm positions below the knee joints with
an infusion of sterile physiological saline solution. Round
dental burrs 0.6 and 1.2 mm in diameter rotating at a speed of
160 rpm were used, powered by a micromotor for implant
surgery. Two implants of the same alloy in a limb were
anteroposterior-bicortically inserted and not positioned inside the medullary cavity parallel to the longitudinal axis of
the tibia. The four alloys were divided into two groups,
namely, the Ti alloy (Ti6Al4V and Ti15Zr4Nb4Ta)
group and the other alloy (316L and CoCrMo) group.
Dierent alloys of the same group were implanted separately
into the left or right tibia. The incised skin was closed by
mattress suture using a surgical silk suture needle. At three
days after implantation, the rats were given an antibiotic. The
rats conditions were checked everyday up to the rst 2
weeks after the operation and then once every 3 days
thereafter. Five Wistar rats for each implantation period per
group (total: 40 rats) were used. A radiograph of the rat tibia
with the Ti15Zr4Nb4Ta alloy implant at 12 weeks after
implantation is shown in Fig. 1.
The left and right tibiae were removed at 6, 12, 24, and 48
weeks after implantation. The bones were fully washed in a
physiological saline solution. The bone tissue was xed in a
10% neutral phosphate-buered formalin solution for more
than two weeks. The procedures for the preparation of the
undecalcied and decalcied bone tissue sections are shown

Knee joint

Fig. 1 Radiograph of Ti15Zr4Nb4Ta alloy implant at 12 weeks after

implantation.

Fixation in 10% formalin

(a)

(b)
Decalcification

Dehydration

Removal and dehydration

Resin

Im
Bone
Bone

Cutting

Immersed in paraffin wax

Paraffin

Slicing of resin block

Resin

Bone
Im

100 m

Slide

Bone

Paraffin wax

Removal of paraffin
wax and dehydration

Polishing
Im:Implant

Bone

Staining

Fig. 2 Schematic diagram of preparation of microscopic sections.

(a) Undecalcied section, (b) decalcied section.

in Fig. 2. In the undecalcied bone section [Fig. 2(a)], the

bone tissue was dehydrated and defatted with ethanol (70%,
80% and 90% overnight, and 100% for 4 d at room
temperature), xylene (for 3 d at room temperature) and
100% acetone (for 3 d at room temperature) successively.
The bone tissue was then immersed in resin (0steobet I and
0steobet II) for 7 d at 4
C, and embedded in fresh resin
(0steobet) for 3 d at 35
C successively. The bone tissues
embedded in the resin were crosscut using a diamond blade
(Microtom, SP1600, Lieca Co., Ltd.) to a thickness of
approximately 100 mm at the longitudinal center of the metal
implant, the position approximately 300 mm upward from the
center, and the position approximately 300 mm downward

1612

Y. Okazaki et al.
Table 2
Subject

Relationship between various stains and dyed nishes.

Osteoid

Calcied bone

Nuclear

Bright eld

Reddish
purple

Colorless
Light brown

Reddish
purple

Fluorescence

Red

Yellowish green
green

Red

Giemsa

Blue

Light
blue

Reddish
purple

Toluidine blue

Blue

Light
blue

Blue

Red

Purple

Stains

Connective tissue

Villanueva

Hematoxylin and eosin

Light
red

Azan-Malloy

Blue

from the center (transverse sectioning). The sliced resin block

was then polished to a thickness below 40 mm using waterproof emery paper from #600 to #4000 grit, followed by
staining using toluidine blue (TB) and Villanueva stains. The
decalcied bone section was used to observe the bone tissue
reactions at a high amplication rate. For decalcied bone
sectioning [Fig. 2(b)], after xation, another bone tissue was
decalcied using a 5% formalin solution containing 5%
formic acid (pH 2:3, 10% formalin: 10% formic acid =
1:1) at 4
C for 3 d. After decalcifying, the bones were
neutralized in a 5% aqueous sodium sulfate solution for 24 h,
and then washed in running water for 24 h. Thereafter, the
bone tissue was carefully cut using a blade parallel to the
length of the tibia, so as to divide the implant into two
sections along its vertical axis. Then, the implant was
removed. The bone tissue was dehydrated and defatted with
ethanol (70, 70, 90, 100, 100 and 100% for 1 h at room
temperature) and xylene (I, II and III for 1 h at room
temperature) successively. The bone tissue was immersed in
paran wax (I, II and III for 1 h at room temperature)
successively. The bone tissue containing the penetrated
paran wax was placed in a cooled metallic mold and the
melted fresh paran wax was then poured into it. The
specimens embedded in the paran wax were sliced to a
thickness of approximately 4 mm using a microcutting
machine. More than twenty slices were obtained from the
center part of the vertical axis of the metal implant. After
removing the paran wax and dehydration, these sliced
sections were stained using hematoxylin eosin (HE) and
Azan-Malloy (AM) stains to observe bone remodeling and
connective tissues. The bone tissues newly formed around the
metal implant were histologically examined using an optical
micrograph.
In an animal implantation test at the Japan Food Research
Laboratory, CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta
alloys were implanted. After ve weeks of feeding, male
Wister/ST rats (purchased from Japan SLC, Inc.) were used
at 15 weeks of age (weight: 380  14 g). The animals were
anesthetized with sodium pentobarbital at a dose of 40 mg/
kg, and the femur and tibia on the right and left sides were
shaved, sterilized and incised using a scalpel. A hole with a
diameter of 1.2 mm was made in the right femur using an
electric trimmer, and an alloy was inserted into the hole. In

Red

Dark
red

the left femur and both tibiae, the same alloy was implanted
in the same manner. At the end of each implantation period
(4, 12, 24 and 48 weeks), the animals were sacriced under
general anesthesia. Three to ve Wistar rats for each
implantation period per alloy specimen (total: 48 rats) were
used. Bones including metal implants were excised and xed
in formalin, dehydrated in ethanol and acetone, and embedded in polyester resin. The bone tissues embedded in the resin
were sliced using a diamond blade parallel to the length of the
femur or tibia, so as to divide the metal implant into two
sections along its vertical axis (vertical sectioning). The
sliced resin section was then polished to approximately 70 to
80 mm using an electric le, followed by staining using the
Villanueva stain to discriminate the osteoid from the calcied
bone. The toluijine blue and Giemsa stains were used for the
histologial analysis of the bone tissue. The osteoid formation
rate was measured under a uorescent microscope using
specimens subjected to Villanueva staining. The relationship
between various stains and dyed nishes is summarized in
Table 2. The body weight of the rats used in this study
linearly increased up to 24 weeks, but negligibly increased at
48 weeks after implantation, as shown in Fig. 3.
2.3 Morphometry
A schematic illustration for estimating the morphometrical
parameters of the new bone in the transverse and vertical
undecalcied bone sections is shown in Fig. 4. The new bone
tissues that formed around the surface of the metal implant
were compared using the following four parameters: (1) new
bone formation rate (%) = (total length of new bone formed
around implant)/(surrounding length of implant existing in
bone marrow)  100; (2) bone contact rate (%) = (total
length in direct contact with implant)/(surrounding length of
implant in bone marrow)  100; (3) osteoid formation rate
(%) using the Villanueva stain = [(total area of osteoid)/
total area of new bone (osteoid plus calcied bone)]  100;
and (4) new bone thickness = (total area of new bone)/(total
length of new bone formed around metal implant). The new
bone thickness in the vertical section [Fig. 4(b)] was
calculated using the following formula: [length of new bone
minus root of {(length of new bone)2 minus 16(area of new
bone)}]/4. These parameters were measured in the bone
sections under a microscope with an image analysis system

Osteocompatibility of Stainless Steel, CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta Alloy Implants in Rat Bone Tissue

Body Weight of Rat

w /g

800

(a)

1613

(b)

700
600

Implant
500

Implant

400

Bone
marrow

Bone
marrow

300

New bone

200

: Nine-week-old male Wister / ST rat

: Six-week-old male Wister rat

100
0

10

20

30

40

Fig. 5 Optical micrograph of new bone formed surrounding Ti15Zr

4Nb4Ta alloy implant at 12 weeks after implantation. (a) Transverse
section of undecalcied bone stained using toluidine blue, (b) vertical
section of undecalcied bone stained using Villanueva stain.

50

Implantation Period, t / week

12 weeks

4 weeks

24 weeks

48 weeks

(a)
Changes in body weight after implantation in rat.

(a)

New bone
Bone formation
area
Bone
contact
area

Implant

Osteoid
Thickness

(b)
Implant
New bone
Surrounding
length
Bone contact
area

Osteoid
Calcified bone

(b)

Thickness
Calcified bone

Implant

Fig. 3

Fig. 4 Schematic illustration for estimating morphometrical parameters of

new bone in transverse (a) and vertical (b) undecalcied bone sections.

(Quantimet 500+, Leica Co., Ltd.) and by the analysis of an

optical micrograph using Mac Scope (Mitani Shoji). The
means and standard deviations were calculated from the
results obtained for the three to ve rats (n 3 to 5).
3.

(c)

Experimental Results

3.1 Histological analysis

Figure 5 shows the optical micrograph of the new bone
formed surrounding the Ti15Zr4Nb4Ta alloy implant
using the undecalcied bone sections stained with the
toluidine blue and Villanueva stains at 12 weeks after
implantation. The Ti15Zr4Nb4Ta alloy implant was
completely surrounded by the new bone in both the transverse (a) and vertical (b) sections. The new bones formed on
the CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta alloy
implants stained using various stains are compared in Fig. 6.
The new bones were well formed around all the alloy
implants in the bone marrow. Neither the resorption of bone
nor abnormalities such as the presence of foreign-body giant
cells and inltration of inammatory cells were observed in
all the alloy implants throughout the implantation period. In
the new bone-metal interface, thin osteoid layers were
observed in all the alloy implants. The osteoid layers around

Bone
marrow

50 m

Fig. 6 Comparison of new bones formed on CoCrMo, Ti6Al4V and

Ti15Zr4Nb4Ta alloy implants (vertical undecalcied section). (a) Co
CrMo alloy implant stained using Giemsa stain, (b) Ti6Al4V alloy
implant stained using Villanueva stain (bright eld), (c) Ti15Zr4Nb
4Ta alloy implant stained using toluidine blue stain.

the CoCrMo and Ti15Zr4Nb4Ta alloy implants stained

using the Villanueva stain are compared in Fig. 7. The
osteoid layers formed around the CoCrMo alloy implants
were slightly thicker than those formed around the Ti6Al
4V and Ti15Zr4Nb4Ta alloy implants. A tendency

1614

Y. Okazaki et al.

(a)

(b)

Remodeling

50 m

(b)

(a)

Osteoblasts

Bone
marrow
(c)

Implant

50 m

(d)

Implant
(f)

50 m

(e)

Bone
marrow
: osteoid
Fig. 7 Osteoid observed in new bone-CoCrMo alloy implant interface
stained using Villanueva stain. (a), (c), (e) Bright eld, (b), (d), (f)
uorescence, (a), (b) 4 weeks after implantation, and (c), (d), (e), (f) 12
weeks after implantation.

(a)

50 m

Fig. 8 Bone remodeling in new bone-implant interface in the transverse

undecalcied sections stained using toluidine blue stain. (a) New boneCoCrMo alloy interface at 6 weeks after implantation, (b) new bone-Ti
15Zr4Nb4Ta alloy interface at 12 weeks after implantation.

similar to that in the vertical section was observed in the

transverse undecalcied sections with the 316L stainless
steel, CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta alloy
implants. The bone remodeling around the new bone-metal
implant interface observed in the transverse undecalcied
sections is shown in Fig. 8. A normal bone remodeling was
observed in the bone-metal interface, and osteoblasts capable
of dierentiating into a new bone tissue were lined on the
implant side in the new bone-metal implant interface. The
new bone formed around the metal implant developed into a
calcied bone consisting of lamellar structure with increasing
implantation period. Many osteocytes were observed in the
lamellar bone tissue. The decalcied bone tissues stained
using the AM and HE stains are shown in Figs. 9(b) to (e). A
capsulated brous connective tissue was observed in the Co
CrMo alloy section at 48 weeks after long-term implanta-

(d)

Implant

Implant
site

Osteoclast

(b)

Implant site

(e)
Osteoblasts
Implant
site

Osteocytes
Bone
marrow

(c) Implant site

Fibrous connective
tissues

Lamellar calcified
bone
50 m

Fig. 9 Undecalcied stained using toluidine

blue (a) and decalcied bone tissue [(b)(e)]
stained using AM [(b)(d)] and HE [(e)] stains.
(a)(d) CoCrMo alloy at 48 weeks after
implantation, (c) polarized optical micrograph,
(e) Ti15Zr4Nb4Ta alloy at 12 weeks after
implantation.

Osteocompatibility of Stainless Steel, CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta Alloy Implants in Rat Bone Tissue

3.2 Osteocompatibility
Osteocompatibilities obtained using the four morphometrical parameters for 316L stainless steel, CoCrMo, Ti
6Al4V and Ti15Zr4Nb4Ta alloy implants are compared
in Figs. 10 to 13. The new bone formation rates in both the
transverse and vertical sections were more than 90% in the
early implantation period for all the alloy implants (Fig. 10).
Thereafter, no marked changes were observed in any
observation period. The bone contact rates in the vertical

120

(a)
100
Transverse section

Bone Formation Rate (%)

60

60

40

40

20

10

20

30

Transverse section
Vertical section

20
50 0
120

40

120
(c)

100

80

80

60

60

40

20

30

40

50

20
10

20

30

40

50

20

t / m

60

40

40

140

10

20

30

40

(c)

120

al
Vertic

sectio

80

60

20

(b)

100

e sectio

ers
Transv

80

20
0
50
140

Transverse section
10

100

80

80

20

30

40

50

(d)

120

Vertical section

100

Vertical section

60
Transverse
section

40
20
0

10

20

30

40

Transverse section

40
20
50
0

10

20

30

40

50

Implantation Period, t / week

Fig. 12 Comparison of new bone thicknesses for 316L stainless steel (a),
CoCrMo (b), Ti6Al4V (c), and Ti15Zr4Nb4Ta (d) alloy implants.

30

(a)

(c)

20

20

10

10

0
0
30

10

20

30

40

50

Vertical section

10

20

30

40

50

Implantation Period, t / week

(b)
20
10
0

10

20

30

40

50

Implantation Period, t / week

40

Transverse section
Vertical section
0

10
(d)

100

100

30

80

120

60

(b)

100

80

140
(a)

120

Osteoid Formation Rate (%)

120

140

New Bone Thickness

tion, as shown in Figs. 9(a) to (d). This capsulated brous

connective tissue was also observed in the 316L stainless
steel section at 48 weeks after implantation. Many osteoclasts
were observed at the interface between the brous connective
tissue and lamellar bone tissue. Osteoblasts and osteocytes
were observed in the decalcied bone section of the Ti15Zr
4Nb4Ta alloy implant stained using the HE stain at 12
weeks after implantation, as shown in Fig. 9(e).

1615

Transverse section
Vertical section
0

10

20

30

40

50

Fig. 13 Comparison of osteiod formation rates for CoCrMo (a), Ti6Al

4V (b), and Ti15Zr4Nb4Ta (c) alloy implants.

Implantation Period, t / week

Fig. 10 Comparison of new bone formation rates for 316L stainless steel
(a), CoCrMo (b), Ti6Al4V (c), and Ti15Zr4Nb4Ta (a) alloy
implants.

Bone Contact Rate (%)

100 (a)

100 (b)

80

80

60

60
Transverse section

40
20
0

10

20

30

40

40
50

20

80

80

60

60

40

10

20

40

Transverse section
Vertical section
0

30

40

50

100 (d)

100 (c)

20

Transverse section
Vertical section

10

20

30

40

50

20

Transverse section
Vertical section
0

10

20

30

40

50

Implantation Period, t / week

Fig. 11 Comparison of bone contact rates for 316L stainless steel (a), Co
CrMo (b), Ti6Al4V (c), and Ti15Zr4Nb4Ta (d) alloy implants.

section tended to be higher than that in the transverse section

(Fig. 11). The bone contact rates in the vertical sections of
CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta alloy implants at 4 weeks after implantation were 40  12, 67  12
and 59  12%, respectively. A signicant dierence in bone
contact rate at 4 weeks after implantation was observed
between the CoCrMo and two Ti alloy implants (p <
0:05). At 12 weeks after implantation, the values were
59  11, 72  10 and 64  11%, respectively. These values
gradually increased to approximately more than 75% at 24
weeks after implantation, and thereafter, remained almost
unchanged. Statistical analysis showed no signicant dierence in bone contact rate between the Ti6Al4V and Ti
15Zr4Nb4Ta alloy implants at p 0:05. The thicknesses
of the new bones formed around the alloy implants are
compared in Fig. 12. The new bone thickness in the vertical
section tended to be higher than that in the transverse section.
The new bone thicknesses in the vertical sections of CoCr
Mo, Ti6Al4V and Ti15Zr4Nb4Ta alloy implants were
approximately 60 mm at 4 weeks after implantation. No
dierence in new bone thickness between the Ti6Al4V and
Ti15Zr4Nb4Ta alloy implants was observed. The osteoid

1616

Y. Okazaki et al.

formation rates of the new bones formed around the alloy

implants are compared in Fig. 13. The osteoid formation
rates were approximately 11  6% at 4 weeks after implantation in the CoCrMo alloy implant. In the Ti15Zr4Nb
4Ta alloy implant, the osteoid formation rates were 10  4,
6  3, 6  2, and 3  1% at 4, 12, 24 and 48 weeks after
implantation, respectively. In contrast, in the CoCrMo and
Ti6Al4V alloy implants, osteoid formation rates slightly
increased from 24 up to 48 weeks after implantation, and the
values were 9  6% and 12  8% at 48 weeks, respectively.
Thus, the four morphometrical parameters obtained in rat
implantation are valuable for the evaluation of osteocompatibility of biomaterials.
4.

Discussion

For long-term implantation, the investigation of osteocompatibility of the metal implants is an important method. It
was claried that the four morphometrical parameters,
namely, new bone formation rate, bone contact rate, new
bone thickness and osteoid formation rate, were important for
the evaluation of osteocompatibility of biomaterials. In view
of animal protection in recent years, histological and
morphometrical analyses using rats are important for the
evaluation of osteocompatibility of biomaterials. The interface between the newly formed bone around the implant and
metal implant is typically loose in the decalcifed bone
section, when the metal implant is pushed out during the
preparation. On the other hand, undecalcied bone sections,
embedded in resin and cut including the metal, are advantageous in the determination of new bone-material interface.
AM staining is a typical method of stain selective staining
of collagen bers using aniline blue, one of the brous
connective tissues. In the early stage of implantation, the
osteoid was stained using aniline blue on the implant side of
the new bone, and the calcied bone was stained in red on the
outside. This tendency was consistent with that in the case of
using TB, in which a new bone was stained light blue on the
outside and deeply stained in the interface with the implant.
The brous connective tissue was observed around the 316L
stainless steel and CoCrMo alloy implants at 48 weeks
after implantation. This result was consistent with the fact
that the stainless steel and CoCrMo alloy have been
classied in the group of metals encapsulated by the brous
connective tissue (capsule type).6) The bone contact rate of
the Ti6Al4V alloy implant tended to be slightly lower than
that of the Ti15Zr4Nb4Ta alloy implant at 48 weeks after
long-term implantation. The bone contact rate of the Ti6Al
4V alloy implant is lower than that of the commercially pure
Ti implant.7,8)
Metals from orthopaedic implants are released by various
mechanisms, including corrosion and mechanically accelerated electrochemical processes such as stress corrosion and
corrosion fatigue.9) The toxic eects of metals released from
prosthetic implants have been reviewed.10) Stainless steel and
CoCrMo and Ti6Al4V alloys having cytotoxic elements, such as Co, Cr, Ni and V, have become a subject of
discussion. To compare the release properties of various
metal elements in vivo, trace quantities of metals accumulating in the bone tissue after implantation into the rat tibia for

up to 48 weeks have been examined.11) The Fe concentration

in the bone tissue with the 316L stainless steel implant was
relatively high, and it rapidly increased up to 12 weeks after
implantation and then decreased thereafter. The Ni concentration in the bone tissue with the 316L stainless steel implant
increased up to 6 weeks after implantation and then gradually
decreased thereafter. On the other hand, the Co concentration
in the tibia tissue with the CoCrMo alloy implant was low,
and it increased up to 24 weeks and slightly decreased at 48
weeks after implantation. The Cr concentration tended to be
higher than the Co concentration. This Cr concentration
linearly increased up to 12 weeks and then decreased toward
48 weeks in the tibia tissues with the 316L stainless steel or
CoCrMo alloy implant. Minute quantities of Ti, Al and V
in the tibia tissues with the Ti6Al4V implant were
observed. The Ti concentration in the tibia tissues with the
Ti15Zr4Nb4Ta implant was lower than that in the tibia
tissues with the Ti6Al4V implant. The Zr, Nb and Ta
concentrations were also very low. In the present study, the
rate of direct bone contact with the two Ti alloys was
signicantly higher than that with the 316L stainless steel and
CoCrMo alloy in the early stage of implantation. These
ndings are considered to reect the in vivo results mentioned
above.
It has been reported that of the 70 metals in the periodic
table, only Zr and Ti support osteoblast growth and
osteointegration.12) Ti, Zr, Nb and Ta also have a considerably superior corrosion resistance. A new Ti alloy, Ti
15Zr4Nb4Ta alloy, without known cytotoxic metal elements such as V has been developed. The relative growth
ratio of murine osteoblastic MC3T3-E1 cells is estimated
using the following formula: (average number of cells per
dish after 4 d incubation)/(average number of cells in the
control). The relative growth ratio of the MC3T3-E1
(1:08  0:02) cells with the Ti15Zr4Nb4Ta alloy is
slightly higher than that of MC3T3-E1 cells with the Ti
6Al4V alloy (1.0).13,14) In this study, the osteoid formation
rate of the Ti15Zr4Nb4Ta alloy implant slightly decreased with increasing implantation period, compared to those of
Ti6Al4V and CoCrMo alloy implants. Metal releases of
Co and Cr as major components of CoCrMo alloy, and V
as the minor component of Ti6Al4V alloy might be
responsible for the eects of these alloys on osteocompatibility.
Anodic polarization is enhanced when Zr, Nb, Ta or Pd is
added because the resultant ZrO2 , Nb2 O5 , Ta2 O5 and PdO
strengthen the TiO2 passive lm that forms on Ti alloy.15)
The quantity of Ti released from the Ti15Zr4Nb4Ta alloy
is lower than that released from the Ti6Al4V alloy in
simulated body uids.16) The quantity of (Zr+Nb+Ta)
released is considerably lower than that of (Al+V) released.
An acceptable level of biological response is expected when
the Ti15Zr4Nb4Ta alloy is used appropriately, because it
consists of biocompatible elements and has excellent
biocompatibility, corrosion resistance, microstructure, and
mechanical and fatigue properties.1726) The facilities required for melting, forging and working of the Ti15Zr4Ta
4Nb alloy are the same as those required for such processes of
the Ti6Al4V alloy. Bone plates, hip screws, intramedullary
xation and articial hip joint implants are fabricated using

Osteocompatibility of Stainless Steel, CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta Alloy Implants in Rat Bone Tissue

1617

the same conventional manufacturing processes used for the

Ti6Al4V alloy.24) The increase in the incidence of allergy
and the necessity for prolonged use require implants with a
minimal metal release rate. Therefore, the Ti15Zr4Ta4Nb
alloy with a low metal release rate is considered advantageous for long-term surgical implantation.

rate, new bone thickness and osteoid formation rate,

suggested to be good markers of osteocompatibility for the
investigation of various metal implants.

5.

1) G. L. Darimont, R. Cloots, E. Seidel and R. Lerrand: Biomaterials 23

(2002) 25692575.
2) Y. T. Sul, C. B. Johansson, K. Roser and T. Albrektsson: Biomaterials
23 (2002) 18091817.
3) C. J. Oosterbos, H. C. Vogely, M. W. Nijhof, A. Fleer, A. J. Verbout,
A. J. Tonino and W. J. Dhert: J. Biomed. Mater. Res. 60 (2002) 339
347.
4) S. Vercaigne, J. G. Wolke, I. Naert and J. A. Jansen: Clin. Oral Implants
Res. 11 (2000) 314324.
5) C. J. Ivano, C. Hallgren, G. Widmark, L. Sennerby and A.
Wennerberg: Clin. Oral Implants Res. 12 (2001) 128134.
6) S. G. Steinemann: Evaluation of Biomaterials. eds. G. D. Winter, J. L.
Leray and K. de Goot, (Wiley, Chichester, 1980) pp. 134.
7) C. B. Johansson, L. Sennerby and T. Albrektsson: Int. J. Oral
Maxillofac Implant 6 (1991) 437441.
8) C. B. Johansson, C. H. Han, A. Wennerberg and T. Albrektsson: Int. J.
Oral Maxillofac Implant 13 (1991) 315321.
9) J. J. Jacobs, C. Silverton, N. J. Hallab, A. K. Skipor, L. Patterson, J.
Black and J. O. Galante: Clin. Orthop. Related Res. 358 (1999) 173
180.
10) K. L. Wapner: Clin. Orthop. Related Res. 271 (1991) 1220.
11) Y. Okazaki, E. Gotoh, T. Manabe and K. Kobayashi: Biomaterials 25
(2004) 59135920.
12) S. G. Steinemann: Materials for Medical Engineering, eds. H.
Stallforth and P. Revell, Euromat 99, Volume 2, (Wiley-VCH,
Weinheim, 1999), pp. 199203.
13) Y. Okazaki and Y. Ito: Adv. Eng. Mater. 2 (2002) 278281.
14) Y. Okazaki: Curr. Opini. Solid State and Mater. Sci. 5 (2001) 4553.
15) Y. Okazaki, T. Tateishi and Y. Ito: Mater. Trans., JIM 38 (1997) 7884.
16) Y. Okazaki and E. Gotoh: Biomaterials 26 (2005) 1121.
17) Y. Okazaki, Y. Ito, A. Ito and T. Tateishi: Mater. Trans., JIM 34 (1993)
12171222.
18) Y. Okazaki, A. Ito, T. Tateishi and Y. Ito: Mater. Trans., JIM 35 (1994)
5866.
19) A. Ito, Y. Okazaki, T. Tateishi and Y. Ito: J. Biomed. Mater. Res. 29
(1995) 893900.
20) Y. Okazaki, Y. Ito, A. Ito and T. Tateishi: Medical Applications of
Titanium and Its Alloys: The Material and Biological Issues, ASTM
STP 1272, eds. S. A. Brown and J. E. Lemons (ASTM 1996) pp. 4559.
21) Y. Okazaki, S. Rao, T. Tateishi and Y. Ito: Mater. Sci. Eng. A 243
(1998) 250256.
22) Y. Okazaki, S. Rao, Y. Ito and T. Tateishi: Biomaterials 19 (1998)
11971215.
23) Y. Okazaki and E. Nishimura: Mater. Trans., JIM 41 (2000) 1247
1255.
24) Y. Okazaki and E. Gotoh: Mater. Trans., JIM 43 (2002) 29432948.
25) Y. Okazaki and E. Gotoh: Mater. Trans., JIM 43 (2002) 29492955.
26) Y. Okazaki: Mater. Trans., JIM 43 (2002) 31343141.

Conclusions

Although thin osteoid layers in the new bone-metal

implant interface were observed in all 316L stainless steel,
CoCrMo, Ti6Al4V and Ti15Zr4Nb4Ta alloy implants, the new bone was well formed around all the alloy
implants in the bone marrow of the rat femur and tibia up to
48 weeks. Neither the resorption of bone nor inammatory
reactions such as that the presence of foreign-body giant cells
and inltration of inammatory cells were evident in the
histological examination of these implants. A normal bone
remodeling was observed in the new bone-metal interface,
and osteoblasts capable of dierentiating into a new bone
tissue were lined on the implant side in the new bone-metal
implant interface. Many osteocytes were observed in the
lamellar bone tissue. In the decalcied bone tissues stained
using the Azan-Malloy stain, a capsulated brous connective
tissue was observed in the 316L stainless steel and CoCr
Mo alloy implants at 48 weeks after long-term implantation.
Many osteoclasts were observed at the interface between the
brous connective tissue and lamellar bone tissue. The new
bone formed around all the alloy implants developed into a
calcied bone consisting of lamellar structure with increasing
implantation period. The bone formation rates around all the
alloy implants were markedly high, approximately more than
90% at 4 weeks after implantation, and thereafter, no
remarkable change was observed. The bone contact rate
and new bone thickness in the vertical section tended to be
higher than those in the transverse section. In the early stage
of implantation (412 weeks), the bone contact rate of Ti
alloy implant was signicantly higher than that of 316L
stainless steel and CoCrMo alloy implants. In the late stage
of implantation (2448 weeks), the osteoid formation rates of
CoCrMo and Ti6Al4V alloy implants tended to increase, but not signicantly. Signicant dierences in the
bone morphometrical parameters, suggesting osteocompatibility, were detected, although pathological ndings were not
evident. Histological examinations of undecalcied sections
were essential for conrming the interface between the newly
formed bone and the metal implant, and morphometrical
parameters, namely, new bone formation rate, bone contact

REFERENCES